Comparative Biochemistry and Physiology Part B 133 (2002) 597–607 1096-4959/02/$ - see front matter  2002 Elsevier Science Inc. All rights reserved. PII: S1096-4959 Ž 02 . 00144-6 Absence of global genomic cytosine methylation pattern erasure during medaka (Oryzias latipes) early embryo development  Ronald B. Walter *, Hai-Ying Li , Gabriel W. Intano , Steven Kazianis , Christi A. Walter a, a b a b,c Department of Chemistry and Biochemistry, Southwest Texas State University, 419 Centennial Hall, 601 University Drive, a San Marcos, TX 78666-4616, USA Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, TX, USA b South Texas Veteran’s Health Care System, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA c Received 22 July 2002; received in revised form 30 July 2002; accepted 2 August 2002 Abstract Two techniques were used to analyze global genomic 5-methyl cytosine methylation at CCGG sites of medaka embryo DNA. DNA was labeled by incorporation of microinjected radiolabeled deoxynucleotide into one-cell embryos. After Hpa II or Msp I digestion the radiolabeled DNA was fractionated in agarose gels and the distribution of label quantified throughout each sample lane to detect differences in fragment distribution. Alternately isolated DNA was digested with Hpa II or Msp I and the resulting generated termini end-labeled. The end-labeled digestion products were then analyzed for fragment distribution after gel fractionation. These techniques proved to be extremely sensitive, allowing comparison of genomic DNA methylation values from as few as 640 fish cells. The data suggest that in medaka embryos the vast majority ()90%) of genomic DNA is methylated at CCGG sites. Furthermore, these data support the conclusion that the extent of methylation at these sites does not change or changes very little during embryogenesis (from 16 cells to the hatchling). These data argue against active demethylation, or loss of methylation patterns by dilution, during the developmental stages between the one cell zygote and gastrulation. From a comparative viewpoint, these data may indicate that mammals and fishes methylate and demethylate their genomes in very different manners during development.  2002 Elsevier Science Inc. All rights reserved. Keywords: Medaka; Fish; Methylcytosine; Genomic DNA; Methylation patterns; Microinjection; Embryogenesis; Restriction analyses 1. Introduction During mammalian embryonic development changes in methylation patterns occur at specific loci resulting in modulation of gene transcriptional activity (Bird, 1984; Sano et al., 1984; Cedar, 1988). It has also been shown that overall DNA methylation is essential for normal embryonic development and expression of select alleles  Contribution to a special issue of CBP on Comparative Functional Genomics. *Corresponding author. q1-512-245-0357; fax: q1-512- 245-1922. E-mail address: RWalter@swt.edu (R.B. Walter). (Shemer et al., 1990; Li et al., 1992, 1993). In some cases genetic traits exhibit a parental depend- ency for expression in progeny. In these situations (termed genetic imprinting) expression of the trait is dependent on receiving an allele from a given parent (Cattanach et al., 1979; McGowan et al., 1989; Chaillet et al., 1991; Stoger et al., 1993). In germ cells, the extent of parental genomic meth- ylation is reduced upon fertilization of the embryo, but are thought to become restored during game- togenesis (Howlett and Reik, 1991; Kafri et al., 1992). These types of studies have led to the suggestion that methylation patterns are intimately associated with genetic imprinting phenomena.